Use of PACAP for the treatment of viral infections in aquatic organisms

ABSTRACT

The present invention relates to the use of pituitary adenylate cyclase activating polypeptide (PACAP) in the treatment of viral diseases and infectious diseases caused by viruses in aquatic organisms. PACAP, alone or combined with an antiviral molecule, demonstrated its effectiveness by increasing the survival of fish or crustaceans infected by viruses when it was administered orally, by injection or by immersion baths. Furthermore, it was observed that treated organisms keep or increase its weight as compared to infected and non-treated organisms. PACAP or PACAP-containing combinations decreased the viral load in tissues and organs susceptible to viral infections as it was determined by RT-PCR.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 13/989,921,filed May 28, 2013, which is a U.S. National Phase of, and Applicantsclaim priority from, International Application No. PCT/CU2011/000008,filed Nov. 30, 2011, and Cuban Patent Application No. 2010-0233, filedDec. 1, 2010, all of which are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The present invention relates to the field of aquatic biotechnology,particularly with the use of PACAP and PACAP-containing compositions fortreating viral infections or infectious diseases caused by viruses inaquatic organisms. It also relates to the use of PACAP in combinationwith antivirals to treat infections caused by virus.

BACKGROUND OF THE INVENTION

The annual contribution of the aquatic species culture to the totalproduction of fish and shellfish has increased in recent times. However,higher densities imply that these animals live in stress conditions and,in general, they are more susceptible to infections. Disease outbreakscaused by pathogens in aquaculture result in large economic losses andeven sometimes produce the removal of certain culture from a specificregion.

In salmonids, the infestations caused by the infectious pancreaticnecrosis virus (IPNV), the viral hemorrhagic septicemia virus (VHSV),the infectious salmon anemia virus (ISAV) and the infectioushematopoietic necrosis virus (IHNV) are the main causes of diseaseoutbreaks in their culture (Marroqui et al. (2008) Antiviral Research80: 332-338). For example, it is estimated that Chilean salmonproduction for the year-end 2010 will fall by 38.7% (from 403 000 tonnesin 2009 until 245 000 tonnes in 2010) as a result of mortality caused byISAV.

Moreover, the crustaceans are one of the most important economic sectorsin global aquaculture, contributing over 10 billion annually. Theshrimps in culture are susceptible to a wide variety of pathogensincluding viruses. It is estimated than in the mid-90 approximately 40%of world production (equivalent to 3 billion U.S. dollars) was lost as aconsequence of diseases. Five viruses are the main contributors to theselosses: the white spot syndrome virus (WSSV), the yellow head virus(YHV), the taura syndrome virus (TSV), the infectious hematopoieticnecrosis virus (IHNNV) and the monodon baculovirus (MBV) (reviewed byJohnson et al. (2008) Vaccine 26: 4885-4992).

Bivalves are an important part of the global shellfish production. Theseorganisms, having the characteristic of being filter feeders, areimportant virus reservoirs; thus their culture may face epizootics whichthreat its production. It has been reported mass mortalities of adult'soysters of Crassostrea angulata associated with viral infections similarto iridovirus. In addition, other viruses of Herpesviridae,Papovaviridae, Togaviridae, Retroviridae, Reoviridae, Birnaviridae andPicornaviridae families are capable to infect bivalve's cultures.However, due to the absence of cell lines derived from shellfish and thelimiting existing molecular tools for these organisms, bivalve virologyis still a primitive science based primarily on morphological studiesand few experimental studies (reviewed by T. Renault and B. Novoa (2004)Aquat. Living Resour. 17: 397-409).

Antiviral drugs are chemical compounds used to treat infections causedby viruses. The first experimental antivirals were discovered in theearly 60's. These were developed based on the “trial” and “error”methodology. However, after the mid 80's, the scene changed dramaticallyand in recent years many novel antiviral drugs have been developed andregistered, mostly for the treatment of the human immunodeficiency virus(HIV). However, since these compounds are not always effective or welltolerated, there are many aspects to be improved. Additional reasons torefine the design and application of these drugs are the emergence ofviral resistance or side effects associated with them (De Clercq (2002)Nature Reviews Drug Discovery 1: 13-25). The antiviral drug design hasas main targets viral proteins or host cell proteins. The first strategyleads to compounds more specific and less toxic, but with a narrowerspectrum of antiviral activity and a greater chance of developingresistance. The second strategy results in discovering antiviralcompounds with a broader spectrum of activity, less likely to developresistance but higher probability to produce cellular toxicity. Thestrategy of choice depends on the nature of the virus and the potentialtargets in the virus or in the host cells (De Clercq (2002) NatureReviews Drug Discovery 1:13-25).

Ribavirin is a broad-spectrum antiviral compound with activity against awide range of DNA and RNA viruses. It is a nucleoside analogue, whichafter intracellular phosphorylation, becomes a competitive inhibitor ofinosine monophosphate dehydrogenase (IMPDH), a cellular enzyme involvedin the synthesis of guanosine monophosphate (GMP) (Graci y Cameron,(2006) Reviews in Medical Virology 16: 49-63; Parker, (2005) VirusResearch 107: 165-171). In addition to the inhibition of this enzyme,there are other three mechanisms which have been proposed to explain theantiviral activity of this compound: by direct inhibition of viral RNApolymerase (Toltzis et al. (1988) Antimicrobial Agents and Chemotherapy32: 492-497), the inhibition of the cap addition at the 5′ end of viralmessenger RNA (mRNA) (Goswami et al. (1979) Biochemical and BiophysicalResearch Communications 89: 830-836) and the accumulation of mutations(Graci y Cameron, (2002) Virology 298: 175-180).

Up to now, there are not specific antiviral drugs approved for thetreatment of viral diseases in aquatic organisms. In Chile, a subsidiaryof Diagnotec SA named Andromaco, developed a new antiviral compoundcalled VIROTOP for the treatment of viral diseases in fish. Thisantiviral is pending for approval by the “Servicio Agricola y Ganadero”(SAG). However, a significant number of compounds have been evaluated invitro and in vivo (Hudson et al. (1988) Antiviral Research 9:379-385;Jasher et al. (2000) Antiviral Research 45: 9-17; Kamei and Aoki, (2007)Archives of Virology 152: 861-869; LaPatra et al. (1998) Fish andShellfish Immunology 8: 435-446; Mas et al. (2006) Antiviral Research72: 107-115; Micol et al. (2005) Antiviral Research 66: 129-136; Moya etal. (2000) Antiviral Research 48: 125-130), showing various degrees ofeffectiveness.

The administration of a ribavirin analog,5-ethynyl-1-β-D-ribofuranosylimidazole-carboxamide (EICAR) has beenevaluated in vivo in larvae of salmon coho (Oncorhynchus kisutch) andrainbow trout (Oncorhynchus mykiss) experimentally infected with IPNV(Moya et al. (2000) Antiviral Research 48: 125-130). Treatment consistedof daily baths in a solution of 0.4 and 0.8 μg/mL of EICAR for two hoursduring 20 days. The results showed that the survival of the infectedgroups and treated with EICAR was similar to survival in the groups notinfected with the virus. An analysis of viral load in liver and spleenwas performed using the technique of reverse transcription chainreaction polymerase (RT-PCR). This analysis demonstrated a decrease inviral load in animals treated with the antiviral. From this study, itwas concluded that EICAR is an inhibitor of IPNV replication, so thattheir use to reduce viral load and prevent fish mortality is abeneficial tool for increasing crops productivity. However, antiviraltreatment is not useful for breeding selection, because infected andtreated fish still carry the virus and can transmit it to offspring. Onthe other hand, it has observed that one of the damage caused by someviral infections is the weight loss in infected fish (Wolf (1986) Thefish viruses In: Espinosa de los Monteros, J., Labarta, U. (Eds.),Patología en Acuicultura. Industrias gráficas, España, SL, pp. 93-95;Moya et al. (2000) Antiviral Research 48: 125-130).

An additional advantage of the use of antivirals is that this weightloss is much lower in fish treated. This effect was most evident introut than in salmo coho (Moya et al. (2000) Antiviral Research 48:125-130).

Pituitary adenylate cyclase activating polypeptide (PACAP) belongs tothe superfamily of secretin/glucagon/vasoactive intestinal peptide. Thispeptide was first isolated from bovine hypothalamus in 1989. It wasdemonstrated its ability to stimulate the secretion of growth hormonethrough activation of adenylate cyclase and subsequent stimulation ofthe production of adenosine monophosphate (cAMP) (Miyata et al (1989)Biochem Biophys Res Commun 164 567-574). PACAP is a multifunctionalneuropeptide that plays important roles as neurotransmitter,neuromodulator and vasodilator in mammals (Arimura A. (1998) JapaneseJournal of Physiology 48:301-31). It has been demonstrated its functionin cell division regulation, differentiation and cell death (Sherwood etal. (2000) Endocrine Review 21: 619-670). This peptide exists in twodifferent molecular variants: 27 aa (PACAP27) and 38 aa (PACAP38)(Miyata et al. (1990) Biochemical and Biophysical ResearchCommunications 170:643-8). The effects of PACAP are exerted through afamily of three VIP/PACAP receptors that belong to the secretinG-protein-coupled receptor. VPAC-1 and VPAC-2 receptors exhibit similaraffinities for the two neuropeptides, VIP and PACAP, whereas PACAPreceptor (PAC-1) exhibits a higher affinity for PACAP than for VIP(Vaudry et al. (2000) Pharmacol Rev 52: 269-324). PACAP is widelydistributed in the mammalian brain, mainly in the hypothalamus, theparaventricular and dorsamedial nuclei of the thalamus, in the septum,the cerebral cortex, the amygdala, the hippocampus and the cerebellum(Montero et al. (2000) Journal of Molecular Endocrinology 25: 157-168).The most abundant variant in the central nervous system and peripheraltissues is PACAP38. The studies performed in mammals showed the presenceof PACAP also in gonads (Shioda et al. (1994) Endocrinology 135:818-825), adrenal glands (Arimura et al. (1991) Endocrinology 129:2787-2789), parathyroid glands (Vaudry et al. (2000) Pharmacol Rev 2000;52: 269-324), endocrine pancreas (Arimura y Shioda (1995)Neuroendocrinology 16: 53-88) and the gastrointestinal tract (Arimura etal. (1991) Endocrinology 129: 2787-2789; Vaudry et al. (2000) PharmacolRev 52: 269-324; Hannibal et al. (1998) Cell. Tissue. Res. 291: 65-79).

PACAP and its receptors expression in immune cells have been onlypartially elucidated (Gaytan et al. (1994) Cell Tissue Res 276:223-7;Abad et al. (2002) NeurolmmunoModulation 10:177-86). In mammals, it hasbeen observed constitutive expression of VPAC-1 receptor in peripheralblood lymphocytes in humans and mice lymphocytes and macrophages,whereas the expression of VPAC-2 is inducible in these cells. Moreover,it has been observed constitutive expression of PAC-1 receptor in ratperitoneal macrophages and human myelomonocytic cell line THP-1.Additionally, it has been reported PACAP expression in thymocytes,different subtypes of T cells, B cells, splenocytes and lymph nodes inrats (Delgado et al. (2001) J Biol Chem 276:369-80; Pozo et al. (2003)Trends Mol Med 9:211-7).

In fish, PACAP has been described in the central (especiallyhypothalamus, brain and spinal cord) and peripheral nervous system,innervating eyes, pituitary gland, respiratory tract, salivary glands,gastrointestinal tract, reproductive tract, pancreas and urinary tract(Sherwood et al. (2000) Endocrine Review 21: 619-670).

PACAP inhibits the spontaneous apoptosis of thymocytes in rats (Delgado.(1996) Blood 87: 5152-5161). The fact that PACAP controls thymocyteproliferation suggests that this peptide is an important regulator ofthe maturation of immune cells (Delgado. (1996) Blood 87: 5152-5161).

This peptide has an indirect effect over lymphocytes maturation throughthe stimulation of interleukin 6 (IL-6) releases by follicular cells inpituitary. The IL-6 stimulates growth and differentiation of B cells andpromotes the synthesis and secretion of immunoglobulins by these cells(Tatsuno et al. (1991) Endocrinology 129: 1797-1804; Yada et al. (1993)Peptides 14: 235-239). Additionally, PACAP activates and suppress theinflammatory response through the regulation of IL-6 and IL-10 (Martínezet al. (1996) J Immunol 156(11):4128-36; Martínez et al. (1998) JNeuroimmunol 85(2):155-67); Martínez et al. (1998) J Leukoc Biol63(5):591-601). In activated macrophages, PACAP inhibit pro-inflammatorycytokines and stimulates the anti-inflammatory cytokines production,allowing the homeostasis of immune system. Besides, PACAP reduces theexpression of co-stimulatory molecules B7.1/137.2 and subsequentactivation of T helper cells (Th). On the other hand, PACAP inhibit theproduction of IL-6 through its receptor PAC-1 in activated macrophages,suppressing inflammation (Martínez et al. (1998) J Neuroimmunol85(2):155-67; Martínez et al. (1998) J Leukoc Biol. 1998 May;63(5):591-601). The inhibitory action of PACAP over IL-6 transcriptionin response to intense inflammatory stimuli or to intoxication helpstissue protection and immune system homeostasis (Martínez et al. (1998)J Neuroimmunol 85(2):155-67; Martinez et al. (1998) J Leukoc Biol.63(5):591-601). In contrast, PACAP induce the expression of B7.2 andpromotes cellular differentiation to Th2 in non-stimulated macrophages(Delgado y Ganea (2001) Arch Immunol Ther Exp (Warsz) 49(2):10110).

In general, the function of PACAP in modulation of mammal's immunesystem has been only partially elucidated in recent years. These studiesdemonstrated that PACAP regulates both, innate and adaptive immunesystem and modulates pro and anti-inflammatory response. Nevertheless,the existing information about the effect of this peptide on antiviralresponse is scarce. Recently, it was demonstrated in hepatitis B chronicpatients, an increase in plasma PACAP-38 levels once the viremia waseliminated as a consequence of lamivudine treatment (Elefsiniotis et al.(2003) European Journal of Gastroenterology and Hepatology 15:1209-1216). This finding suggests an effect over T cells immune responsewhich results in a biochemical and histological disease remission in thepatient's liver.

In fish, the in vivo studies about PACAP biological function publisheduntil now are mainly related with reproduction (Canosa et al. (2008)American Journal of Physiology (Regul Integr Comp Physiol) 295:1815-21),brain development (Sherwood et al. (2007) Peptides 28:1680-7) andappetite (Matsuda et al. (2005) Peptides 26:1611-6; Maruyama et al.(2006) Peptides 27:1820-6). Recently, it was demonstrated the biologicalfunction of this neuropeptide in growth and development of differentlarval teleost species (Lugo et al. (2008) Journal of Endocrinology197:583-97).

The knowledge about the function of PACAP in the modulation of fishimmune response is limited to studies performed by the inventors'research group. It was demonstrated that recombinant Clarias gariepinusPACAP administration by baths or injection not only promotes growth butstimulates also innate immune parameters (lysozyme, nitric oxide derivedmetabolites and antioxidant defences) and acquired immunity (IgM) inlarvae and juveniles treated (Carpio et al. (2008) Fish and ShellfishImmunology 25:439-45; Lugo et al. (2010) Fish and Shellfish Immunology29:513520). These new properties were described in the patentapplication “Neuropéptidos para el cultivo de organismos acuáticos”(WO2007/059714).

In vertebrates, one of the first defense line against viral infectionsis the type I interferon (IFN) system. This system is activated by viralinduction of type I interferons (IFNα and IFNβ) through the IFN-α/βreceptor which triggers signal transduction mediated by JAK-STAT. Theinduced cytokines produce a cell antiviral state producing theexpression of proteins with antiviral activity such as 2′,5′oligoadenilate synthetase, quinase R and the GTPases Mx (Goodbourn etal. (2000) Journal of General Virology 81: 2341-2364). Recent evidencesestablish that fish have an IFN system similar to mammals (Robertsen(2006) Fish and Shellfish Immunology 20: 172-191; Robertsen (2008) Fishand Shellfish Immunology 25: 351357). The interferons have been clonedin several fish species like zebrafish (Danio rerio), American catfish(Ictalurus punctatus) and Atlantic salmon (Salmo salar) (Altmann et al.(2003) Journal of Virology 77: 1992-2002; Lutfalla et al. (2003) BMCGenomics 4: 29; Robertsen et al. (2003) Journal of Interferon andCitokine Research 23: 601-612; Long et al. (2006) Fish and ShellfishImmunology 21: 42-59). Additionally, it has been identified several IFNregulatory factors, molecules of the JAK-STAT signaling pathway, Mxproteins and other genes stimulated by IFN in different fish species. Itwas reported antiviral activity mediated by Mx proteins in Salmo salarand Paralichtys olivaceus (reviewed by Robertsen (2006) Fish andShellfish Immunology 20: 172-191).

Despite the fact that it has not been discovered IFN like genes incrustaceans yet, it has been observed a negative regulation of STATmolecules (these are molecules which are activated in the IFN responsein vertebrates) in response of WSSV infection. The negative regulationof STAT during a viral infection antagonizes the type I IFN response inmammals (reviewed by Johnson et al. (2008) Vaccine 26: 4885-4992). Inbivalves, the knowledge of antiviral response is even less. There areevidences of antiviral activity in the hemolymph of these organisms andit has been suggested the presence of an IFN like mechanism (Olicarda etal. (2005) Antiviral Research 66(2-3): 147-152; Defer et al., (2009)Aquaculture 293(1-2): 1-7).

In aquaculture, it is of great interest the development of new compoundsor compositions that can be employed in the control of viral infections,due to the damages that they cause in this activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Expression analysis by real time PCR of PAC-1 receptor in headkidney leukocytes from rainbow trout (Oncorhynchus mykiss) treated withpoly I:C (30 μg/mL) or infected with IPNV (multiplicity of infection(m.o.i) of 0.1) at 4, 24 and 48 h from the beginning of the experiment.The leukocytes cultures were treated by duplicated and the expressionlevels were analyzed by qPCR in triplicate. Data are expressed as meanof relative expression of PAC-1 related to the endogenous elongationfactor EF1α±standard deviation (SD). * (p<0.05).

FIG. 2. Expression analysis by real time PCR of PAC-1 receptor in themacrophage cell line from O. mykiss RTS-11 infected in vitro with VHSV(m.o.i of 0.1) at 1, 4 and 8 h from the beginning of the experiment. Theexperiment was done in duplicated and the expression levels wereanalyzed by qPCR in triplicate. Data are expressed as mean of relativeexpression of PAC-1 related to the endogenous elongation factorEF1α±standard deviation (SD). * (p<0.05).

FIG. 3. Expression analysis by real time PCR of VPAC-1 receptor in headkidney leukocytes from rainbow trout (O. mykiss) treated with poly I:C(30 μg/mL) or infected with VHSV (multiplicity of infection, m.o.i of0.1) at 4, 24 and 48 h from the beginning of the experiment. Theleukocytes cultures were treated by duplicated and the expression levelswere analyzed by qPCR in triplicate. Data are expressed as mean ofrelative expression of VPAC-1 related to the endogenous elongationfactor EF1α±standard deviation (SD). * (p<0.05).

FIG. 4A. Expression analysis by real time PCR of PAC-1 receptor in headkidney leukocytes from rainbow trout (O. mykiss) experimentally in vivoinfected with VHSV (100 μL of 1×10⁷ TCID50 (viral titer in culturesupernatant/mL per fish). Five pools of total RNA from head kidney of 5fish were sampled at random at 3, 7 and 10 days after viral infection.The expression levels were analyzed by qPCR in triplicate. Data areexpressed as mean of relative expression of PAC-1 related to theendogenous elongation factor EF1α±standard deviation (SD). * (p<0.05).

FIG. 4B. Expression analysis by real time PCR of PAC-1 receptor inspleen from rainbow trout (O. mykiss) experimentally in vivo infectedwith VHSV (100 μL of 1×10⁷ TCID50 (viral titer in culture supernatant/mLper fish). Five pools of total RNA from spleen of 5 fish were sampled atrandom at 3, 7 and 10 days after viral infection. The expression levelswere analyzed by qPCR in triplicate. Data are expressed as mean ofrelative expression of PAC-1 related to the endogenous elongation factorEF1α±standard deviation (SD). * (p<0.05).

FIG. 5A. Expression analysis by real time PCR of VPAC-1 receptor in headkidney leukocytes from rainbow trout (O. mykiss) experimentally in vivoinfected with VHSV (100 μL of 1×10⁷ TCID50/mL per fish). Five pools oftotal RNA from head kidney of 5 fish were sampled at random at 3, 7 and10 days after viral infection. The expression levels were analyzed byqPCR in triplicate. Data are expressed as mean of relative expression ofVPAC-1 related to the endogenous elongation factor EF1α±standarddeviation (SD). * (p<0.05).

FIG. 5B. Expression analysis by real time PCR of VPAC-1 receptor inspleen from rainbow trout (O. mykiss) experimentally in vivo infectedwith VHSV (100 μL of 1×10⁷ TCID50/mL per fish). Five pools of total RNAfrom spleen of 5 fish were sampled at random at 3, 7 and 10 days afterviral infection. The expression levels were analyzed by qPCR intriplicate. Data are expressed as mean of relative expression of VPAC-1related to the endogenous elongation factor EF1α±standard deviation(SD). * (p<0.05).

FIG. 6. Expression analysis by real time PCR of PACAP in spleenleukocytes from rainbow trout (O. mykiss) experimentally in vivoinfected with VHSV (100 μL of 1×10⁷ TCID50/mL per fish). Five pools oftotal RNA from spleen of 5 fish were sampled at random at 3, 7 and 10days after viral infection. The expression levels were analyzed by qPCRin triplicate. Data are expressed as mean of relative expression ofPACAP related to the endogenous elongation factor EF1α±standarddeviation (SD). * (p<0.05).

FIG. 7A. Expression analysis by real time PCR of the in vitro effect ofPACAP38 over the transcription of Mx protein coding gene in peripheralblood leukocytes of healthy rainbow trout. The effects of PACAPadministration at a concentration of 10⁻¹⁰, 10⁻⁹ and 10⁻⁸ M wasevaluated at 48 h post-treatment. The experiment was repeated 4 times.The leukocytes culture was treated by duplicated and the qPCR wasperformed by triplicate. Data are expressed as mean of relativeexpression of Mx related to the endogenous elongation factorEF1α±standard deviation (SD). * (p<0.05).

FIG. 7B. Expression analysis by real time PCR of the in vitro effect ofPACAP38 over the transcription of Mx protein coding gene in head kidneyof healthy rainbow trout. The effects of PACAP administration at aconcentration of 10⁻¹⁰, 10⁻⁹ and 10⁻⁸ M was evaluated at 48 hpost-treatment. The experiment was repeated 4 times. The leukocytesculture was treated by duplicated and the qPCR was performed bytriplicate. Data are expressed as mean of relative expression of Mxrelated to the endogenous elongation factor EF1α±standard deviation(SD). * (p<0.05).

FIG. 8A. Expression analysis by real time PCR of the in vitro effect ofPACAP38 over the transcription of IFN γ coding gene in peripheral bloodleukocytes of healthy rainbow trout. The effects of PACAP administrationat a concentration of 10⁻¹⁰, 10⁻⁹ and 10⁻⁸ M was evaluated at 48 hpost-treatment. The experiment was repeated 4 times. The leukocytesculture was treated by duplicated and the qPCR was performed bytriplicate. Data are expressed as mean of relative expression of IFN γrelated to the endogenous elongation factor EF1α±standard deviation(SD). * (p<0.05).

FIG. 8B. Expression analysis by real time PCR of the in vitro effect ofPACAP38 over the transcription of IFN γ coding gene of healthy rainbowtrout. The effects of PACAP administration at a concentration of 10⁻¹⁰,10⁻⁹ and 10⁻⁸ M was evaluated at 48 h post-treatment. The experiment wasrepeated 4 times. The leukocytes culture was treated by duplicated andthe qPCR was performed by triplicate. Data are expressed as mean ofrelative expression of IFN γ related to the endogenous elongation factorEF1α±standard deviation (SD). * (p<0.05).

FIG. 9A. Expression analysis by real time PCR of the in vitro effect ofPACAP38 over the transcription of TLR9 coding gene in peripheral bloodleukocytes of healthy rainbow trout. The effects of PACAP administrationat a concentration of 10⁻¹⁰, 10⁻⁹ and 10⁻⁸ M was evaluated at 48 hpost-treatment. The experiment was repeated 4 times. The leukocytesculture was treated by duplicated and the qPCR was performed bytriplicate. Data are expressed as mean of relative expression of TLR9related to the endogenous elongation factor EF1α±standard deviation(SD). * (p<0.05).

FIG. 9B. Expression analysis by real time PCR of the in vitro effect ofPACAP38 over the transcription of TLR9 coding gene in head kidney ofhealthy rainbow trout. The effects of PACAP administration at aconcentration of 10⁻¹⁰, 10⁻⁹ and 10⁻⁸ M was evaluated at 48 hpost-treatment. The experiment was repeated 4 times. The leukocytesculture was treated by duplicated and the qPCR was performed bytriplicate. Data are expressed as mean of relative expression of TLR9related to the endogenous elongation factor EF1α±standard deviation(SD). * (p<0.05).

FIG. 10. In vitro assays to determine the antiviral activity ofPACAP-ribavirin combinations (Rib-PACAP). At 24 h previous to virusinfection, cells were treated with the “antiviral” or the combinationsPACAP-antivirals. Positive control (c+): Cells infected with viruswithout antiviral treatment. Negative control (c−): Cells non-infectedand non-treated. Cells were infected with virus during 30 min to allowadsorption. Afterward, they were washed with PBS and the differenttreatments were added. The inhibition of cytopathic effects (CPE) wasevaluated employing the crystal violet stain (absorbance at 550 nm).

DESCRIPTION OF THE INVENTION

The present invention provides a solution to the above described problemproviding a new alternative to control viral infections in aquaculture,by the use of PACAP in the manufacture of compositions for the treatmentof viral infections and infectious diseases caused by virus in aquaticorganisms.

The term “treatment” in the context of the present invention is relatedto any beneficial effect in disease remission which includesattenuation, reduction or decrease of the pathological development afterthe disease onset.

The term “antiviral”, as it is used in the present invention, includesany molecule defined as antiviral in the previous art or its analogs,functional derivatives or active fragments.

The term “Pituitary Adenylate cyclase Activating Polypeptide (PACAP)”,as it is used in the present invention, includes this molecule in anyvariant (PACAP27 or PACAP38), either isolated from its natural source,or synthetic, or produced by recombinant DNA technology.

In one embodiment of the invention, variants of the neuropeptide PACAPare administered alone or in combination with a known antiviral moleculeto fish, crustaceans and bivalves, by immersion baths with 2-3 daysintervals. As a feed additive, PACAP is also administrated alone or incombination with a known antiviral molecule.

In the present invention, it was demonstrated that PACAP containingcompositions administered by immersion baths, injection or orally wereeffective increasing survival of the aquatic organism infected by virusand treated with them. No decrease in the weight of treated fish wasobserved. In some cases an overall weight increase was obtained ininfected and treated animals as compared to infected and non-treated.The analysis of tissue samples and susceptible organs by RT-PCR showed adecreased in the viral load in the animals infected and treated.

Up to this invention there was no knowledge about whether or not PACAPmodulated the antiviral response in fish, crustaceans and bivalves andthe mechanisms involved. The present invention demonstrates, for thefirst time, both in vitro and in vivo, a relationship between PACAP andthe response to virus in fish, crustaceans and bivalves. Additionally,described for the first time is an unexpected positive effect on PACAPmolecules involved in antiviral response, such as Mx protein, interferongamma (IFN γ) and toll like receptor 9 (TLR9) as well as a differentialregulation of this peptide and its receptors in cells of the immunesystem of fish treated with virus and/or poly I:C, both in vitro and invivo.

The treatment of viral infections in fish, crustaceans and bivalves bythe administration of PACAP or a combination of PACAP with a moleculewith known antiviral action in fish has not been revealed or suggestedpreviously in the state of the art.

In the context of the present invention, administration of PACAP and theantiviral molecule may be simultaneous (in a single formulation),sequential or separate over time. The preparations are administered toaquatic organisms by oral route, injection or immersion baths.

In the present invention, fish, crustaceans and bivalves are treatedwith PACAP and preparations containing antiviral molecules combined withPACAP. Unexpectedly, it was observed that the neuropeptide PACAP hasantiviral activity in fish, crustaceans and bivalves. On the other hand,it was shown that the combination of PACAP with substances with knownantiviral activity produce a higher antiviral response (synergisticeffect) as compared to the PACAP alone, both in vitro and in vivo. Infish, so far, no biological functions have been assigned to PACAP in theantiviral response. Moreover, to date neither PACAP nor another peptidebelonging to the superfamily of secretin and glucagon has been isolatedin shrimp or shellfish. The present knowledge of this peptide in thesetaxonomic groups is only based on experimental evidence of growthstimulation in shrimps by exogenous administration of recombinant fishPACAP (patent application publication number WO2007/059714international). Therefore, until the present invention, no knowledgeabout its possible relationship with response to virus in crustaceansand bivalves existed.

Another object of this invention is a composition for the treatment ofviral and infectious diseases caused by viruses in aquatic organismswhich comprises the “activating polypeptide pituitary adenylate cyclase”(PACAP). In one embodiment of the invention, the PACAP, as part of thecomposition, is obtained by isolation either from its natural source,synthetically or by recombinant DNA technology. It is administeredeither orally, by injection or immersion baths. In one embodiment of theinvention PACAP is a polypeptide sequence isolated from fish.

The present invention also relates to a veterinary combination whichcomprises “activating polypeptide pituitary adenylate cyclase” (PACAP)and an antiviral molecule. In one embodiment of the invention theantiviral molecule is ribavirin or a ribavirin analog. The elements thatare part of the combination, the antiviral molecule and PACAP, areadministered simultaneously, separately or sequentially during the sametreatment. In one embodiment of the invention, the combination is usedin the treatment of viral and infectious diseases caused by viruses inaquatic organisms. In these, the PACAP and antiviral are administeredorally, by injection or immersion baths.

In one embodiment of the invention, PACAP is applied as formulated feedat a concentration of 50-750 mg/kg of feed and the antiviral molecule ata concentration of 100 to 2000 mg/kg of feed. In a second embodiment ofthe invention, the PACAP is applied to fish by injection at aconcentration of 0.1-10 μg per gram of body weight and the antiviral ata concentration of 1-50 μg/g, as part of the veterinary combination. Inanother embodiment of the invention in this combination, the PACAP isapplied to fish or crustaceans by immersion baths at a concentration of50-1000 μg per liter of water and the concentration of the antiviral is100-2000 μg/L.

In the invention, both the PACAP when used as an antiviral, or incombination with a known antiviral molecule is applied to a variety ofaquatic organisms. For example, they include salmonids, penaeid shrimps,and bivalves.

The composition comprising PACAP and the combination with an antiviralmolecule are useful in therapeutic treatment of viral infections causedby viruses of the Herpesviridae, Papovaviridae, Togaviridae,Retroviridae, Reoviridae, Birnaviridae and Picornaviridae families.

In the invention, both the PACAP when used as an antiviral and thecombination with an antiviral molecule, are used to control infectionscaused by IPNV, VHSV and ISAV. Also, they are used in the therapeutictreatment of viral infections caused by WSSV, YHV, TSV, IHNNV and MBV.

It is also part of the present invention a method for the control ofviral infections in aquaculture management comprising the administrationof PACAP or veterinary combinations comprising PACAP to aquaticorganisms in culture.

EXAMPLES Example 1 PAC-1 Receptor Expression in Leukocytes from HeadKidney of Rainbow Trout (O. mykiss) Cells Treated with Poly I:C and IPNVand in the Macrophage Cell Line of Rainbow Trout (O. mykiss) RTS-11Infected with VHSV In Vitro

Juvenile rainbow trout (O. mykiss) was used from 9 to 12 grams and 7months of age, VHSV and IPNV free. Fish were maintained at 14° C. withwater circulation. Food was administered twice a day ad libitum. Thevirus, VHSV (strain 0771) and IPNV (strain Sp) were propagated in therainbow trout gonad cell line RTG-2 (Sena and Rio (1975) Infect Immun11:815-22). Cells were grown in minimal essential medium (MEM,Invitrogen, USA) supplemented with 10% of fetal calf serum (FCS,Invitrogen) containing 100 IU/mL of penicillin and 100 μg/mL ofstreptomycin. The viruses were inoculated into RTG-2 cells grown in MEMwith antibiotics and 2% FCS at 14° C. When extensive cytopathic effectswere observed, the culture supernatants were harvested and centrifugedto remove cellular debris. For the experiments, the clarified culturesupernatants were used. The viral titers were determined in 96-wellplates as reported by Reed and Muench (Reed and Muench (1998) J Hyg27:280-9).

Head kidney leukocytes were isolated from 4 trout following the methoddescribed by Graham and Secombes (Graham and Secombe (1998) Immunology65:293-7). Briefly, anterior kidney was removed aseptically and passedthrough a nylon mesh of 100μ using medium Leibovitz (L-15, Gibco, UK)supplemented with penicillin 100 IU/mL, streptomycin (100 mg/mL),heparin (10 units/mL) and 2% FCS. The resulting cell suspension wascarefully placed on a Percoll gradient to 51%. The ring of cellscorresponding to leukocytes was removed carefully, washed twice in FCScontaining L-15 at 0.1%. The cells were resuspended in L-15 with 5% FCSat a concentration of 5×10⁶ cells per ml and dispensed in 24-well platesat 1 mL per well. The infected leukocytes were exposed to 30 μg/mL depoly I:C (Sigma) or infected with IPNV at a MOI of 0.1 and incubated at14° C. during 4 hours, 24 hours and 48 hours periods.

Finally, total RNA was purified from leukocytes cultures using themethod described by Chomczynski and Sacchi (Chomczynski and Sacchi(1987) Anal. Biochem. 162:156-9) to evaluate the expression levels ofthe PAC-1 receptor on leukocytes untreated and treated with Poly I:C orinfected with viruses. Samples were treated with the RQ1 RNase-freeDNase (Promega), in order to remove genomic DNA present in the samples.For the synthesis of complementary DNA (cDNA) was used a commercialreagent SuperScript III (Invitrogen) based on the reverse transcriptase.For quantitative PCR reactions (qPCR) was used a mixture of PCR fromcommercial sources: Power SYBR Green PCR Master Mix (AppliedBiosystems). The qPCR results were normalized against a constitutiveendogenous gene expression, specifically elongation factor 1α (EF 1α)and were performed in triplicate. The results were expressed as2^(−ΔCt), where ΔCt is equal to the remainder of the gene Ct value minusthe assessed value of Ct normalizer gene (EF 1α).

The result was that levels of PAC-1 was over-expressed in culturedleukocytes treated with poly I:C at 4, 24 and 48 hours of theexperiment, suggesting that PAC-1 receptor is involved in the responseto virus in fish, taking into account that poly I:C is a molecule thatis structurally the same as double-stranded RNA, an effect similar to aviral infection (many viruses have RNA genomes). These results are shownin FIG. 1. Poly I:C interacts with toll-like receptor-3 (TLR3) that isexpressed in intracellular compartments of B cells and dendritic cells.The TLR-3 is stimulated by viruses in nature.

Moreover, the expression levels of PAC-1 became detectable values ofleukocytes in untreated cultures to undetectable levels in cultures ofleukocytes infected with IPNV at 4, 24 and 48 hours after infection,confirming involvement of this receptor that specifically binds to PACAPin the antiviral response in fish (FIG. 1). The differences observedbetween the effects of viral mimetic poly I:C and that of IPNV could beassociated with a specific effect of the virus in infected cells,probably to their advantage.

Additionally, the expression levels of PAC-1 were evaluated in themacrophage cell line trout (O. mykiss) RTS-11 infected with VHSV, theprocess of viral infection of the line RTS-11 was similar to thatdescribed above for the RTG-2 line. Similarly, a down regulation of theexpression levels of PAC-1 to 4 and 8 was observed days after infectionwith VHSV (FIG. 2).

Example 2 Expression of VPAC-1 Receptor in Leukocytes from AnteriorKidney of Rainbow Trout (Oncorhynchus mykiss) Cells Treated with PolyI:C and VHSV

To assess the levels of expression of VPAC-1 receptor an experimentaldesign similar to the one described in Example 1 was performed.

The result was that the levels of VPAC-1 is over-expressed in culturedleukocytes treated with poly I:C at 4, 24 and 48 hours of theexperiment, suggesting that VPAC-1 receptor as the PAC-1 is involved inthe response to virus in fish, taking into account that poly I:C isconsidered a viral mimetic molecule (FIG. 3).

Moreover, the expression levels of VPAC-1 were undetectable in culturesof leukocytes infected with VHSV at 4, 24 and 48 hours after infection,confirming an involvement of this receptor that binds PACAP and VIP withthe same affinity in the antiviral response in fish (FIG. 3).

As observed with PAC-1 receptor in cultured anterior kidney leukocytesinfected with IPNV, the observed differences in the regulation of VPAC-1by treatment of leukocytes in vitro with poly I:C and the infection VHSVcould be associated with a specific effect of the virus as a biologicalentity complete in infected cells, probably to their advantage.

Example 3 Expression of PACAP and PAC-1 Receptors and VPAC-1 inLeukocytes from Spleen and Anterior Kidney of Rainbow Trout(Oncorhynchus mykiss) Infected with VHSV In Vivo

VHSV (strain 0771) was propagated in the cell line RTG-2 (Sena and Rio(1975) Infect Immun 11:815-22). In summary, the viruses were inoculatedin RTG-2 cell line grown in MEM with antibiotics (100 IU/mL penicillinand 100 μg/mL streptomycin) and 2% FCS at 14 C. When extensivecytopathic effects were observed, the culture supernatants wereharvested and centrifuged to remove cellular debris. For infection oftrout were used clarified culture supernatants. The viral titers weredetermined in 96-well plates as reported by Reed and Muench (Reed andMuench (1998) J Hyg 27:280-9).

Juvenile rainbow trout (O. mykiss) was used from 9 to 12 grams and 7months old and VHSV and IPNV free. Fish were maintained at 14 C withwater circulation. Food was administered twice a day ad libitum. Twoexperimental groups were formed of 20 trout each group, one group wasinjected with virus solution (100 L of 1×10⁷ TCID50/mL per fish) and theother group (used as negative control) was injected with culture mediumRTG-2 not infected with VHSV.

At days 1, 3, 7 and 10 post-injection, 5 trout were taken at random andwere sacrificed by over-exposure to the anesthetic MS-22. Subsequentlythe kidney was removed and spleen above to proceed with the purificationof total RNA from these organs, according to the method described byChomczynski and Sacchi (Chomczynski and Sacchi (1987) Anal. Biochem.162:156-9). CDNA synthesis and reactions of quantitative PCR performedas described in Example 1.

For the reactions of reverse transcription (RT), five total RNAs fromfive individuals from each experimental group selected at random on day1, 3, 7 and 10 of the experiment were pooled.

The result was that in the spleen, PAC-1 and VPAC-1 receptors areover-expressed at day 3 post-injection compared to negative control. Theexpression levels are lower than the control group at day 10post-injection. In anterior kidney, PAC-1 and VPAC-1 receptors havelower expression levels in the control group at day 1 and 3, where thevalues of VPAC-1 to day 10 undetectable. At day 7 both receptorsincrease their levels of expression compared to negative control group.It was also observed that both receptors have a similar expressionpattern over time after viral infection (FIGS. 4 and 5).

The PACAP expression values at day 1 are lower compared to its negativecontrol. On day 7 these expression values are superior. These resultscorrelate with those of its two receptors VPAC-1 and PAC-1, in the sensethat levels of receptor expression showed a tendency to be lower (day 1)and higher (day 7) compared to negative control group. At day 10 theexpression of PACAP values are lower compared to negative control group(FIGS. 4, 5 and 6).

The variation in expression levels of PACAP and its receptors PAC-1 andVPAC-1 in the spleen and anterior kidney of fish experimentally infectedwith VHSV and IPNV demonstrate the involvement of both receptors in theantiviral response in fish in vivo. This was confirmed in challengeexperiments in which a positive effect of PACAP was observed in theantiviral response in salmonids, increasing the survival of infectedfish, enhancing the immune response to viral infection and clearing thevirus from the organs and fluids capable to perform viral replication.

Example 4 PACAP Effect on Transcription of Proteins Mx, IFN and TLR9 inPeripheral Blood Leukocytes and Anterior Kidney of Healthy Rainbow Trout(Oncorhynchus mykiss)

Juvenile rainbow trout (O. mykiss) was used of approximately 50 grams,VHSV and IPNV free. Fish (n=5) were anesthetized with methanesulfonicacid salt (Sigma, USA) and were removed aseptically, first peripheralblood from the tail vein and then the anterior kidney were isolated fromperipheral blood leukocytes from anterior kidney and trout from 5independently, following the method described by Graham and Secombe(Graham and Secombe (1998) Immunology 65:293-7).

The peripheral blood of each fish was diluted 4 times in half Leibovitz(L-15, Gibco, UK) supplemented with penicillin 100 IU/mL, streptomycin(100 μg/mL), heparin (10 units/mL) and 2% FCS and placed carefully on aPercoll gradient to 51%. The ring of cells corresponding to leukocyteswas removed carefully, washed twice in L-15 containing 0.1% FCS.

The anterior kidney was passed through a nylon mesh of 100 μm Leibovitz(L-15, Gibco, UK) supplemented with penicillin 100 IU/mL, streptomycin(100 μg/mL), heparin (10 units/mL) and 2% FCS. The resulting cellsuspension was diluted 1:4 in the same medium supplemented L-15 andplaced carefully on a Percoll gradient to 51%. The ring of cellscorresponding to leukocytes was removed carefully, washed twice in L-15containing 0.1% FCS.

The cells were suspended in L-15 with 5% FCS at a concentration of 5×106cells per ml and dispensed in 24-well plates at 1 mL per well. Theleukocytes were treated with 3 doses of PACAP38 from Clarias gariepinusobtained by chemical synthesis (10⁻¹⁰ M, 10⁻⁹ M and 10⁻⁸ M) in duplicateand used as negative control untreated leukocytes dispensed induplicate.

The levels of Mx, IFN and TLR9 were evaluated 48 hours after treatment.In order to do so, total RNA was purified from leukocyte culturestreated with PACAP by the method described by Chomczynski and Sacchi(Chomczynski and Sacchi (1987) Anal. Biochem. 162:156-9).

The primers designed to amplify Mx, IFN γ and TLR9 do not discriminatebetween cDNA and genomic DNA, total RNAs were treated purified fromdifferent tissues with a DNA nuclease, specifically with RQ1 RNase-freeDNase (Promega) in order to remove genomic DNA may be present in thesamples.

For the synthesis of cDNA it was used a commercial reagent kit usesSuperScript III reverse transcriptase (Invitrogen). Finally, forquantitative PCR reactions (qPCR) was used a mixture of PCR fromcommercial sources: Power SYBR Green PCR Master Mix (AppliedBiosystems). The qPCR results were normalized against a constitutiveendogenous constitutive expression gene specifically elongation 1α (EF1α) and were performed in triplicate. The results were expressed as2^(−ΔCt), where ΔCt is equal to the remainder of the gene Ct value minusthe assessed value of Ct normalizer gene EF 1α).

The Mx protein levels increase after 48 hours of treatment in culturesof peripheral blood leukocytes treated with 10⁻¹⁰ M PACAP38. A dose of10⁻⁹ M, there is also a stimulatory effect of PACAP38 on thetranscription of Mx proteins relative to negative control, but theexpression levels detected were below levels obtained with the dose of10⁻¹⁰ M. A dose of 10⁻⁸ M did not differ in levels of Mx proteinexpression relative to negative control. These results show a positiveeffect of PACAP38 on the transcription of Mx proteins at lowconcentrations in the range of (10⁻⁹-10⁻¹⁰ M) (FIG. 7 A). In head kidneyleukocytes, the effect of PACAP 38 on the Mx protein is more moderatethan in peripheral blood leukocytes, showing a stimulatory effect at adose of 10⁻⁹ M (FIG. 7B).

Additionally, a stimulatory effect of PACAP38 was observed on theexpression of TLR9, from undetectable levels of TLR9 in culturedperipheral blood leukocytes stimulated with PACAP at detectable levelsin cultures stimulated with PACAP38 in the range (10⁻¹⁰-10⁻⁸ M) Thehighest values were obtained at a dose of 10⁻¹⁰ M (FIG. 8 A). In headkidney leukocytes, as it was previously observed with the Mx proteinexpression, the effect was more moderate. The TLR9 expression levels wasstatistically higher than the negative control at the intermediate doseof 10⁻⁹ M (FIG. 8 B).

IFN γ levels were undetectable in non-treated cultures of peripheralblood leukocytes. The cultures treated with 3 doses of PACAP38, resultedin detectable levels of expression with the highest level at theintermediate dose of 10⁻⁹ M (FIG. 9 A). Moreover, in cultured anteriorkidney leukocytes, it was observed that PACAP38 stimulates thetranscription of IFN γ. The expression levels of IFN γ werestatistically higher than the control group at doses of 10⁻⁹ and 10⁻⁸ M,obtaining the best results in the highest dose of 10⁻⁹ M (FIG. 9 B).

Example 5 In Vitro Effect of the PACAP Administration in Combinationwith Ribavirin on IPNV Infection

Supernatant from cell culture (strain ATCC VR-299) infected with IPNV asisolated viral was used. Viral confirmation was made through a specificPCR. For the in vitro assays, the cell line CHSE-214 (Chinook salmonembryo) (ECACC No 00/F1031) was used. This was maintained at 18° C. inMEM medium supplemented with Eagle salts, 10% fetal bovine serum (FBS),2 mM glutamine and antibiotics.

The viral inoculum was grown at 15° C. and 2% FBS and before the assaysthe viral titer in the culture supernatant (TCID 50) was determined bythe technique of Reed and Muench (Reed and Muench (1938) The AmericanJournal of Hygiene 27: 493-497). From viral inoculum a dilution of 0.1moi was prepared. Plates of 96-wells were seeded with CHSE-214maintained at 18° C. in MEM (Eagle's salts) supplemented with 10% FBS, 2mM glutamine and antibiotics. Different concentrations of ribavirin and3 different concentrations of PACAP (0.1, 1 and 10 nM) were evaluated.At 24 hours prior to infection, cells were treated with the antiviral orthe PACAP-antiviral combinations, establishing positive controls (cellsinfected with virus without the antiviral treatment) and negative(uninfected and untreated cells, and uninfected cells treated with theantiviral or with different doses of PACAP). Cells were infected withthe viral inoculum for a period of 30 minutes of adsorption. Then thecells were washed with PBS. The treatments were diluted in MEMsupplemented with 2% FBS and antibiotics and added again. The test wasconcluded once the cytopathic effect (CPE) in the positive control wasobserved. The evaluation was performed by microscopy and by stainingwith crystal violet (550 nm absorbance reading). The CPE in the positivecontrol was observed at 3 days post-infection. The results of the CPEinhibition with respect to untreated and infected control cells,obtained from the absorbance values of crystal violet staining, wereshown in FIG. 10. The analysis of the antiviral activity of ribavirin incombination with PACAP was performed using the program Calcusyn forWindows (Biosoft). The value of the combination index of Chou obtainedshowed synergism between both drugs for the concentration values tested.

Example 6 Effect of Administration of PACAP and PACAP Plus RibavirinCombination in Rainbow Trout (O. mykiss) Experimentally Infected withIPNV

An experiment was conducted to evaluate the effect of administration ofPACAP and PACAP-ribavirin combination in rainbow trout experimentallyinfected with IPNV. Eight experimental groups of 50 fish each at a bodyweight of 1.6±0.4 g were used. The water temperature was kept at 10-12°C.

-   Group 1: untreated fish not exposed to the virus-   Group 2: untreated fish, exposed to the virus-   Group 3: Group treated with ribavirin at a dose of 400 μg/L of    water, exposed to the virus-   Group 4: Group treated with ribavirin at a dose of 800 μg/L of    water, exposed to the virus-   Group 5: Group treated with PACAP at a dose of 100 μg/L of water,    exposed to the virus-   Group 6: Group treated with PACAP at a dose of 200 μg/L of water,    exposed to the virus.-   Group 7: Group treated with the combination 1 (ribavirina 400    μg/L-PACAP 100 μg/L, exposed to the virus.

Group 8: Group treated with the combination 2 (ribavirina 800 μg/L-PACAP200 μg/L exposed to the virus.

The animals were fed twice a day with an amount equivalent to 3% of thetotal corporal weight in each tank. The fish were exposed to the virusby placing them in water at 10-12° C. containing approximately 10⁵plaque forming units (pfu)/mL of IPNV (strain ATCC VR-299) for 2 h. Fishin Groups 1 and 2 were subjected to the same stress treatment withoutthe antiviral compound or without the PACAP-antiviral combination. Thefish in Group 1 received the same procedure with cell culture mediuminstead of virus. The virus identification was performed by RT-PCR fromkidney and spleen, according to the method described by Lopez-Lastra etal. (1994) Journal of Fish Diseases 17: 269-282. Treatments with PACAPor with PACAP-antiviral combination were performed 3 times a week for 20days. The first treatments were performed 2 h after viral infection.Experimental observations were made during the first 20 days oftreatments and 25 days after it concluded. During the experiment (45days) deaths and abnormal behavior of the fish were monitored.

The IPNV infected fish that were not treated showed characteristic signsof infectious pancreatic necrosis from day 6 post-infection. The firstdeath occurred on day 7 post-infection, accompanied by the worsening ofthe signs of the disease in all fish and a high mortality on days 11, 12and 13 post-infection. In fish infected and treated with ribavirin orPACAP the signs of disease were detected on day 8. The groups treatedwith the combination ribavirin-PACAP and exposed to the virus notpresent signs of the disease during the period of the experiment.

The untreated and infected fish recovered appetite on day 15post-infection. Fish treated with the antiviral recovered appetite onday 13, whereas fish treated with PACAP and PACAP-ribavirin combinationsshowed it on day 9 of the experiment. Fish uninfected controls showednormal behavior and there was not mortality in this group during theperiod of the experiment. Cumulative mortality in the untreated fish andIPNV-infected fish was 40 fish of 50 (20% survival). In fish treatedwith ribavirin and infected the survival was 68 and 70% for groups 3 and4, respectively. In fish treated with PACAP and infected the survivalwas 50 and 55% for Groups 5 and 6, respectively. In the groups treatedwith the PACAP-ribavirin combinations the survival was 97 and 99% inGroups 7 and 8, respectively. In tissue samples of dead fish was foundthe presence of IPNV. No virus was detected in samples taken from theuntreated and uninfected groups.

Table 1 shows the average weights at 45 days post-infection. Fishinfected and not treated had a lower weight gain compared to uninfectedfish. This weight loss was offset in the treated groups and more in thegroups treated with PACAP-ribavirin combination.

TABLE 1 Increase in body weight of juvenile rainbow trout during the 45days post-infection. Experimental Initial Increment with respect toGroup Weight (g) Final Weight (g) initial weight (%) 1 1.6 ± 0.4 4.5 ±1.1^(a) 181 2 1.6 ± 0.4 2.7 + 1.2^(b) 69 3 1.6 ± 0.4 3.8 ± 1.1^(c) 137 41.6 ± 0.4 3.7 ± 1.0^(c) 131 5 1.6 ± 0.4 5.0 ± 0.5^(d) 212 6 1.6 ± 0.45.2 ± 0.6^(d) 225 7 1.6 ± 0.4 5.5 ± 0.4^(d) 343 8 1.6 ± 0.4 5.6 ±0.5^(d) 350

An ANOVA followed by a Tukey post test was performed. Different lettersrepresent statistically significant differences.

Example 7 Effect of the Administration of PACAP and the PACAP PlusRibavirin Combination Formulated in Polylactide Acid-Co-Glycolic Acid,to Rainbow Trout (O. mykiss) Experimentally Infected with IPNV

An experiment was conducted to evaluate the effect of administration ofPACAP and PACAP-ribavirin combination contained in polylacticacid-co-glycolic acid (PLGA) to rainbow trout experimentally infectedwith IPNV. Eight experimental groups were performed of 30 fish of 30±4 geach one and were kept in water at 10-12° C.:

-   Group 1: fish injected with PLGA, not exposed to the virus Group 2:    fish injected with PLGA exposed to the virus-   Group 3: Group injected with ribavirin at a dose of 4 μg/g of fish,    exposed to the virus-   Group 4: Group injected with ribavirin at a dose of 8 μg/g of fish,    exposed to the virus-   Group 5: Group PACAP injected at a dose of 0.1 μg/g of fish, exposed    to the virus-   Group 6: Group PACAP injected at a dose of 0.2 μg/g of fish exposed    to the virus-   Group 7: Group injected with the combination 1 (ribavirin 4    μ/g-PACAP 0.1 μg/g), exposed to the virus-   Group 8: group injected with the combination 2 (ribavirin 8    μg/g-PACAP 0.2 μg/g), exposed to the virus

Nanoparticles containing PACAP or PACAP-ribavirin combination, wereadministered by intraperitoneal injection 2 hours after viral infection.Experimental observations were conducted for 30 days. Results onsurvival rates similar to those described in Example 6 were obtained.

Example 8 Effect of Administration of PACAP and the PACAP Plus RibavirinCombination in Atlantic Salmon (Salmo salar) Experimentally Infectedwith IPNV

An experiment was conducted to evaluate the effect of the administrationof PACAP and PACAP-ribavirin combination for salmon (Salmo salar)experimentally infected with IPNV. Eight experimental groups wereperformed of 50 fish and a size of 1.2±0.3 g each one and were kept inwater at 10-12° C.:

-   Group 1: untreated fish not exposed to the virus-   Group 2: untreated fish, exposed to the virus-   Group 3: Group treated with ribavirin at a dose of 400 μg/L of    water, exposed to the virus-   Group 4: Group treated with ribavirin at a dose of 800 μg/L of    water, exposed to the virus-   Group 5: Group treated with PACAP at a dose of 100 μg/L of water,    exposed to the virus-   Group 6: Group treated with PACAP at a dose of 200 μg/L, exposed to    the virus-   Group 7: Group treated with ribavirin 400 μg/L-PACAP 100 μg/L    combination, exposed to the virus-   Group 8: Group treated with ribavirin 800 μg/L-PACAP 200 μg/L    combination, exposed to the virus.

The animals were fed twice a day with an amount equivalent to 3% of thetotal corporal weight in each tank. The fish were exposed to the virusby placing them in water at 10-12° C. containing approximately 10⁵pfu/mL of IPNV (strain ATCC VR-299) for 2 h. Treatments were performed 3times a week for 20 days. Experimental observations were made duringthis period and 25 days after the last treatments. During the 45 days ofthe experiment deaths and abnormal behavior of the fish were monitored.Fish in Groups 1 and 2 were subjected to the same stress produced by thetreatment without the antiviral compound or the combination ofPACAP-antiviral. The fish in Group 1 received the same procedure withcell culture medium instead of the virus. The identification of thevirus was performed by RT-PCR from kidney and spleen, according to themethod described by López-Lastra et al. (1994) Journal of Fish Diseases17: 269-282.

The fish infected with IPNV and not treated showed characteristic signsof infectious pancreatic necrosis from day 6 post-infection. The firstdeath occurred on day 8 post-infection, accompanied by the worsening ofthe signs of the disease in all fish and a high mortality between day 11and 15 post-infection. The percentage of survival in this group was 30%.There was a loss of appetite on days 10 and 11 in all groups infectedwith the virus, with the exception of the groups treated with PACAP,PACAP-ribavirin combination and the uninfected group. In fish infectedand treated with both doses of ribavirin early signs of disease weredetected on day 10 and there was a 70-76% survival for the low and highdoses, respectively. In fish infected and treated with PACAP thesurvival was a 56-60% for the low and high doses, respectively. In thegroup treated with the combination ribavirin-PACAP and exposed to thevirus the survival was a 96-98%. In the uninfected and untreated groupthe percentage of survival was 98%. In tissue samples of dead fish wasfound the presence of IPNV.

Table 2 shows the average weights at 45 days post-infection. Fishinfected and untreated had a lower weight gain compared to uninfectedfish. This weight loss was offset in the treated groups and more in thegroups treated with PACAP and PACAP-ribavirin combinations.

TABLE 2 Increase in body weight of juvenile Atlantic salmon during the45 days post-infection. Experimental Group Initial Weight (g) FinalWeight (g) Increment (%) 1 1.2 ± 0.3 2.5 ± 0.8^(a) 108 2 1.2 ± 0.3 2.0 ±0.4^(b) 67 3 1.2 ± 0.3 2.2 ± 0.4^(c) 83 4 1.2 ± 0.3 2.3 ± 0.5^(c) 92 51.2 ± 0.3 2.4 ± 0.3^(a) 200 6 1.2 ± 0.3 2.8 ± 0.3  233 7 1.2 ± 0.3 3.1 ±0.2  258 8 1.2 ± 0.3 3.4 ± 0.1  283

An ANOVA followed by a Tukey post test was performed. Different lettersrepresent statistically significant differences.

Example 9 Effect of Administration of PACAP and PACAP Plus RibavirinCombination in Atlantic Salmon (Salmo salar) Experimentally Infectedwith ISAV

An experiment was conducted to evaluate the effect of the administrationof PACAP and PACAP-ribavirin combination for salmon (Salmo salar)experimentally infected with ISAV. Eight experimental groups wereperformed of 25 fish and 50±10 g each one. The groups were kept in seawater at 10-12° C.

-   Group 1: Fish untreated, not exposed to the ISA virus-   Group 2: untreated fish, exposed to the ISA virus-   Group 3: Group treated with ribavirin at a dose of 400 μg/L of    water, exposed to the virus-   Group 4: Group treated with ribavirin at a dose of 800 μg/L of    water, exposed to the virus-   Group 5: Group treated with PACAP at a dose of 100 μg/L of water,    exposed to the virus-   Group 6: Group treated with PACAP at a dose of 200 μg/L of water,    exposed to the virus-   Group 7: Group treated with the combination of ribavirin 400    μg/L-PACAP 100 μg/L, exposed to the virus-   Group 8: Group treated with the combination of ribavirin 800    μg/L-PACAP 200 μg/L, exposed to the virus

To obtain sufficient virus for the challenge experiments were culturedSHK-1 cells and infected with the strain Glesvaer (access No. AJ012285,Krossoy et al. (1999) Journal of Virology 73: 2136-2142) in 175 cm²flasks for 5 days.

Fish were exposed to the virus by cohabitation with fish injectedintraperitoneally with 0.3 mL of culture medium containing approximately10⁴ pfu/mL of ISAV. Group 1: fish that received an equal volume ofculture medium. The % of mortality in Group 1 (untreated, not exposed tothe virus ISA) was 4% and in Group 2 (exposed to virus and untreated)was 92%. In the Groups 3 and 4, mortality was 52 and 36%, respectivelywhile in Groups 5 and 6 was 60 and 72%, respectively. In Groups 7 and 8treated with PACAP-ribavirin combination the % of mortality was 8 and4%, respectively. In tissue samples of dead fish was found the presenceof ISAV. No virus was detected in samples from untreated and uninfectedgroup.

Example 10 Effect of the Administration of PACAP and PACAP-amantadineCombination on Rainbow Trout (O. mykiss) Experimentally Infected withIHNV

An experiment was conducted to evaluate the effect of the administrationof PACAP and PACAP-amantidine combination in rainbow troutexperimentally infected with IHNV. Eight experimental groups wereperformed of 50 fish of 50±5 g each one and were kept in water at 10-12°C.

-   Group 1: untreated fish not exposed to the virus-   Group 2: untreated fish, exposed to the virus-   Group 3: Group treated with amantadine at a dose of 400 μg/L of    water, exposed to the virus-   Group 4: Group treated with amantadine at a dose of 800 μg/L of    water, exposed to the virus-   Group 5: Group treated with PACAP at a dose of 100 μg/L of water,    exposed to the virus-   Group 6: Group treated with PACAP at a dose of 200 μg/L of water    exposed to the virus-   Group 7: Group treated with the combination 1 (amantidina 400    μg/L-PACAP 100 μg/L exposed to the virus-   Group 8: Group treated with the combination 2 (amantidina 800    μg/L-PACAP 200 μg/L exposed to the virus

The experimental conditions were similar to the experiments described inthe previous examples. The fish were challenged by immersion with5.9×10³ pfu/ml virus in 20 L of water with aeration for 1 hour. As aresult of the experiment a survival of a 26% was observed in theuntreated and infected fish with IHNV. In fish treated with amantadineand infected with IHNV the survival was 46%. In fish treated with PACAPand infected with IHNV the survival was 36%. In the groups treated withthe PACAP-amantidine combination the survival was 96-98%.

Example 11 Effect of the Administration of PACAP and the PACAP PlusRibavirin Combination on Pacific White Shrimp (Litopenaeus vannamei)Experimentally Infected with the Virus of White Spot Syndrome (WSSV)

An experiment was conducted to evaluate the effect of the administrationof PACAP and PACAP-ribavirin combination on Pacific white shrimpLitopenaeus vannamei experimentally infected with WSSV. Fourexperimental groups were formed of 50 shrimp of 5±0.5 g each one andwere kept in seawater at 28° C.:

The shrimp were fed twice a day with an amount equivalent to 8% of thetotal biomass per group. The experimental groups were:

-   Group 1: untreated, not exposed to the virus WSSV-   Group 2: untreated, exposed to the virus WSSV-   Group 3: Treated with ribavirin, exposed to the virus WSSV-   Group 4: Treated with PACAP, exposed to the virus WSSV-   Group 5: treated with the combination ribavirin-PACAP, exposed to    the virus WSSV For the isolation and viral spread was used    Orconectes limosus crab. WSSV was purified from freshly extracted    hemolymph by sucrose gradient centrifugation as described by Van    Hulten et al in 2001 (Van Hulten et al. (2001) Virology 285: 22833).

The viral samples were stored at −80° C. until use.

In order to minimize the natural routes of infection with WSSV and toachieve reproducible protocol the immersion via was used to induce theexperimental viral infection. Prior to the viral infection the viraltiter and the amount required to cause 75% mortality was determined. Todo that the shrimp were experimentally infected with different dilutionsof the virus during an incubation period of 7 hours.

Following the viral infection, shrimps were washed with seawater free ofvirus and placed in new containers according to the design of theexperimental groups described above.

The treatments were administered 3 times per week for 30 days at a doseof (500 μg of antiviral/L of water) or (500 μg of antiviral plus PACAP200 □μg/L of water), post-infection. For the group treated with PACAPthe used dose was of 200 μg/L of water. Shrimp in Groups 1 and 2 weresubjected to the same stress of treatments administration. During the 30days of the experiment deaths in each experimental group were monitored.

The % of mortality in the Group 2 (untreated, exposed to the virus) was60% in Group 1 (untreated, not exposed to the virus) was 0% in Group 3(treated with ribavirin, exposed virus) mortality was 16%, while thePACAP treated group mortality was 26%. In the group treated withPACAP-ribavirin combination % mortality was 2%. In tissue samples ofdead shrimp were detected WSSV by RT-PCR.

Example 12 Effect of Administration of PACAP and the PACAP PlusRibavirin Combination in Oysters of the Species Pecten maximusExperimentally Infected with IPNV

An experiment was conducted to evaluate the effect of administration ofPACAP and PACAP-ribavirin combination in oysters of the species Pectenmaximus experimentally infected with IPNV. Eight experimental groupswere performed of 30 adult oysters each one and were kept in 250 L offiltered sea water at 10° C. No virus was detected in hepatopancreassamples collected prior to the start of the experiment. The experimentalgroups were:

-   Group 1: untreated, not exposed to the virus-   Group 2: untreated, exposed to the virus-   Group 3: Group treated with ribavirin at a dose of 400 μg/L of    water, exposed to the virus-   Group 4: Group treated with ribavirin at a dose of 800 μg/L of    water, exposed to the virus-   Group 5: Group treated with PACAP at a dose of 100 μg/L of water,    exposed to the virus-   Group 6: Group treated with PACAP at a dose of 200 μg/L of water    exposed to the virus-   Group 7: Group treated with the combination 1 (ribavirina 400    μg/L-PACAP 100 μg/L exposed to the virus-   Group 8: Group treated with the combination 2 (ribavirina 800    μg/L-PACAP 200 μg/L exposed to the virus

Treatments were performed 3 times a week for 20 days. Oysters werechallenged by exposure to 25 L of water containing 10⁷ TCID50 mL⁻¹ intanks of 80 L. After 6 h the water was replaced at a slow flow of 1L/min and at 12 h the flow was increased to 3 L/min.

Two weeks after challenge hepatopancreas samples were taken anddetermined the viral titer. The results showed a decrease in viral loadin animals treated with ribavirin and PACAP. This decrease was greaterin the groups treated with the combination (Table 3).

TABLE 3 IPNV average viral titer (log₁₀TCID50 g⁻¹) in the hepatopancreasof oysters two weeks after the challenge. Different letters indicatesignificant differences. Groups Viral Titer 1 0 2 8.1^(a) 3 5.4^(b) 44.3^(b) 5 6.5^(c) 6 5.8^(c) 7 2.0^(d) 8 1.5^(d)

The invention claimed is:
 1. A method for the treatment of viralinfections and infectious diseases caused by viruses in aquaticorganisms comprising administering Pituitary Adenylate CyclaseActivating Polypeptide (PACAP) to aquatic organisms, wherein aquaticorganisms are selected from the group consisting of fish, crustaceans,and bivalves.
 2. The method of claim 1 wherein the PACAP is obtained a)by isolation from its natural source, b) synthetically, or c) byrecombinant DNA technology.
 3. The method of claim 2 wherein the PACAPis isolated from fish.
 4. The method of claim 1 wherein the fish aresalmonids, and the crustaceans are penaeid shrimps.
 5. The method ofclaim 1 wherein the PACAP is used in the therapeutic treatment of viralinfections caused by viruses, wherein the viruses are selected from thegroup consisting of Herpesviridae, Papovaviridae, Togaviridae,Retroviridae, Reoviridae, Birnavirida, and Picornaviridae families. 6.The method of claim 1 further comprising administering an antiviralmolecule selected from the group consisting of ribavirin, a ribavirinanalog, 5-ethynyl-1-β-D-ribofuranosylimidazole-carboxamide, andamantadine.
 7. The method of claim 6 wherein the PACAP and the antiviralmolecule are administered simultaneously, separately, or sequentiallyduring the same treatment.
 8. The method of claim 6 wherein the PACAPand the antiviral molecule are administered orally, by injection or byimmersion bath.
 9. The method of claim 8 wherein the PACAP and antiviralmolecule are part of a formulated feed, wherein the PACAP is at aconcentration of 50-750 μg/kg of feed and the antiviral molecule is at aconcentration of 100-2000 μg/kg of feed.
 10. The method of claim 8wherein the PACAP and antiviral molecule are administered by injection,wherein the PACAP is at a concentration of 0.1-10 μg/gbw (gram of bodyweight) and the antiviral molecule at a concentration of 1-50 μg/gbw.11. The method of claim 8 wherein the PACAP and antiviral molecule areadministered by immersion bath, wherein the PACAP is at a concentrationof 50-1000 μg/L of water and the antiviral molecule is at aconcentration of 100-2000 μg/L of water.